Metabolic systems are often the first networks to respond to environmental changes, and the ability to monitor metabolite dynamics is key for understanding these cellular responses. Because ...monitoring metabolome changes is experimentally tedious and demanding, dynamic data on time scales from seconds to hours are scarce. Here we describe real-time metabolome profiling by direct injection of living bacteria, yeast or mammalian cells into a high-resolution mass spectrometer, which enables automated monitoring of about 300 compounds in 15-30-s cycles over several hours. We observed accumulation of energetically costly biomass metabolites in Escherichia coli in carbon starvation-induced stationary phase, as well as the rapid use of these metabolites upon growth resumption. By combining real-time metabolome profiling with modeling and inhibitor experiments, we obtained evidence for switch-like feedback inhibition in amino acid biosynthesis and for control of substrate availability through the preferential use of the metabolically cheaper one-step salvaging pathway over costly ten-step de novo purine biosynthesis during growth resumption.
Direct injection of samples on high-resolving mass spectrometers is an effective way to maximize analytical throughput and yet allow analyte discrimination in complex samples by mass-to-charge ratio. ...We present a platform of flow injection electrospray–time-of-flight mass spectrometry to profile small molecules in >1400 biological extracts per day at native mass resolution. We comprehensively benchmark the performance with more than 5000 injections of chemically defined standards and Escherichia coli cellular extracts obtained from miniscale cultivations. For at least 90% of tested compounds, we attain a linear response over 3 decades of concentration, interday coefficient of variation of <20%, and a mass accuracy of <0.001 amu. In polar Escherichia coli fractions, we reproducibly detected >1500 distinct ions in each mode. The accurate mass and correlation analysis enabled one to assign with good confidence 400–800 ions to electrospray derivatives of metabolites listed in the genome-wide reconstruction of Escherichia coli metabolism. Hence, we attain a coverage of about one-quarter of the total number of compounds listed in the reconstruction. Finally, we present an exemplary screen with Escherichia coli deletion mutants to show the exquisite capacity of the platform to identify lesions in primary metabolism at high throughputs.
Our understanding of metabolism is limited by a lack of knowledge about the functions of many enzymes. Here, we develop a high-throughput mass spectrometry approach to comprehensively profile ...proteins for in vitro enzymatic activity. Overexpressed or purified proteins are incubated in a supplemented metabolome extract containing hundreds of biologically relevant candidate substrates, and accumulating and depleting metabolites are determined by nontargeted mass spectrometry. By combining chemometrics and database approaches, we established an automated pipeline for unbiased annotation of the functions of novel enzymes. In screening all 1,275 functionally uncharacterized Escherichia coli proteins, we discovered 241 potential novel enzymes, 12 of which we experimentally validated. Our high-throughput in vitro metabolomics method is generally applicable to any purified protein or crude cell lysate of its overexpression host and enables performing up to 1,200 nontargeted enzyme assays per working day.
Metabolism is one of the best‐understood cellular processes whose network topology of enzymatic reactions is determined by an organism's genome. The influence of genes on metabolite levels, however, ...remains largely unknown, particularly for the many genes encoding non‐enzymatic proteins. Serendipitously, genomewide association studies explore the relationship between genetic variants and metabolite levels, but a comprehensive interaction network has remained elusive even for the simplest single‐celled organisms. Here, we systematically mapped the association between > 3,800 single‐gene deletions in the bacterium Escherichia coli and relative concentrations of > 7,000 intracellular metabolite ions. Beyond expected metabolic changes in the proximity to abolished enzyme activities, the association map reveals a largely unknown landscape of gene–metabolite interactions that are not represented in metabolic models. Therefore, the map provides a unique resource for assessing the genetic basis of metabolic changes and conversely hypothesizing metabolic consequences of genetic alterations. We illustrate this by predicting metabolism‐related functions of 72 so far not annotated genes and by identifying key genes mediating the cellular response to environmental perturbations.
Synopsis
The metabolome of > 3,800 single Escherichia coli gene deletion mutants is analyzed. The obtained gene–metabolite interaction map allows predicting orphan gene functions and interpreting a cell's response to environmental perturbations.
Systematic metabolome profiling of > 3,800 single‐gene deletions in the bacterium E. coli charts an empirical map of gene–metabolite associations.
Metabolic changes in deletion mutants reveal a largely unknown landscape of gene–metabolite interactions that are not represented in metabolic models.
Metabolic fingerprint and gene–gene similarity reveals the potential functionality of 72 orphan genes.
The broad applicability of metabolome profiling in gene deletions for interpreting metabolome changes to external perturbations is demonstrated.
The metabolome of > 3,800 single Escherichia coli gene deletion mutants is analyzed. The obtained gene–metabolite interaction map allows predicting orphan gene functions and interpreting a cell's response to environmental perturbations.
To counteract oxidative stress and reactive oxygen species (ROS), bacteria evolved various mechanisms, primarily reducing ROS through antioxidant systems that utilize cofactor NADPH. Cells must ...stabilize NADPH levels by increasing flux through replenishing metabolic pathways like pentose phosphate (PP) pathway. Here, we investigate the mechanism enabling the rapid increase in NADPH supply by exposing Escherichia coli to hydrogen peroxide and quantifying the immediate metabolite dynamics. To systematically infer active regulatory interactions governing this response, we evaluated ensembles of kinetic models of glycolysis and PP pathway, each with different regulation mechanisms. Besides the known inactivation of glyceraldehyde 3-phosphate dehydrogenase by ROS, we reveal the important allosteric inhibition of the first PP pathway enzyme by NADPH. This NADPH feedback inhibition maintains a below maximum-capacity PP pathway flux under non-stress conditions. Relieving this inhibition instantly increases PP pathway flux upon oxidative stress. We demonstrate that reducing cells' capacity to rapidly reroute their flux through the PP pathway increases their oxidative stress sensitivity.
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•Characterization of the immediate metabolic response of E. coli to oxidative stress•Development of a high-performance ensemble modeling computational pipeline•Identification of NADPH feedback inhibition on G6PDH as key regulatory interaction•Cells without reserve flux capacity in PP pathway are sensitive to oxidative stress
Christodoulou et al. describe the immediate metabolic response of E. coli to oxidative stress and reveal the key interactions that implement it. Combining quantitative metabolite dynamics with a high-performing ensemble modeling computational pipeline, they reveal the importance of a small molecule–protein interaction in the rapid cellular response. This interaction acts as a valve, maintaining a reserve of flux, which can be used rapidly when cells are exposed to stress. Cells without such a reserve capacity are more sensitive to oxidative stress.
The thioredoxin-1 (Trx1) system is an important contributor to cellular redox balance and is a sensor of energy and glucose metabolism. Here we show critical c-Myc-dependent activation of the Trx1 ...system during thymocyte and peripheral T-cell proliferation, but repression during T-cell quiescence. Deletion of thioredoxin reductase-1 (Txnrd1) prevents expansion the CD4
CD8
thymocyte population, whereas Txnrd1 deletion in CD4
CD8
thymocytes does not affect further maturation and peripheral homeostasis of αβT cells. However, Txnrd1 is critical for expansion of the activated T-cell population during viral and parasite infection. Metabolomics show that TrxR1 is essential for the last step of nucleotide biosynthesis by donating reducing equivalents to ribonucleotide reductase. Impaired availability of 2'-deoxyribonucleotides induces the DNA damage response and cell cycle arrest of Txnrd1-deficient T cells. These results uncover a pivotal function of the Trx1 system in metabolic reprogramming of thymic and peripheral T cells and provide a rationale for targeting Txnrd1 in T-cell leukemia.
The overall composition of the mammalian intestinal microbiota varies between individuals: within each individual there are differences along the length of the intestinal tract related to host ...nutrition, intestinal motility and secretions. Mucus is a highly regenerative protective lubricant glycoprotein sheet secreted by host intestinal goblet cells; the inner mucus layer is nearly sterile. Here we show that the outer mucus of the large intestine forms a unique microbial niche with distinct communities, including bacteria without specialized mucolytic capability. Bacterial species present in the mucus show differential proliferation and resource utilization compared with the same species in the intestinal lumen, with high recovery of bioavailable iron and consumption of epithelial-derived carbon sources according to their genome-encoded metabolic repertoire. Functional competition for existence in this intimate layer is likely to be a major determinant of microbiota composition and microbial molecular exchange with the host.
Although the mammalian microbiota is well contained within the intestine, it profoundly shapes development and metabolism of almost every host organ. We questioned the range and depth of microbial ...metabolite penetration into the host, and how this is modulated by intestinal immunity. Chemically identical microbial and host metabolites were distinguished by stable isotope tracing from 13C-labeled live non-replicating Escherichia coli, differentiating 12C host isotopes with high-resolution mass spectrometry. Hundreds of endogenous microbial compounds penetrated 23 host tissues and fluids after intestinal exposure: subsequent 12C host metabolome signatures included lipidemia, reduced glycolysis, and inflammation. Penetrant bacterial metabolites from the small intestine were rapidly cleared into the urine, whereas induced antibodies curtailed microbial metabolite exposure by accelerating intestinal bacterial transit into the colon where metabolite transport mechanisms are limiting. Pervasive penetration of microbial molecules can cause extensive host tissue responses: these are limited by immune and non-immune intestinal mucosal adaptations to the microbiota.
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•Metabolites from mutualistic bacteria broadly penetrate host tissues and organs•Bacterial metabolites induce widespread host metabolic and immunological responses•The small intestine is highly susceptible to host-microbial metabolomic exchange•Secretory immunoglobulins accelerate microbial clearance from the small intestine
Bacteria-derived metabolites pervade the mammalian host, shaping immunity and metabolism. Using stable isotope tracing, Uchimura and colleagues profile the scope and depth of host tissue penetration by bacterial metabolites. Extensive host immune and metabolic responses to microbial metabolite penetration are constrained by secretory antibodies that limit microbial small-intestinal dwell time.
The redox cofactor NADPH is required as a reducing equivalent in about 100 anabolic reactions throughout metabolism. To ensure fitness under all conditions, the demand is fulfilled by a few ...dehydrogenases in central carbon metabolism that reduce NADP
with electrons derived from the catabolism of nutrients. In the case of
growing on glucose, quantitative flux analyses indicate that NADPH production largely exceeds biosynthetic needs, suggesting a hitherto unknown mechanism for NADPH balancing. We investigated the role of the four malic enzymes present in
that could bring about a metabolic cycle for transhydrogenation of NADPH into NADH. Using quantitative
C metabolic flux analysis, we found that isoform YtsJ alone contributes to NADPH balancing
and demonstrated relevant NADPH-oxidizing activity by YtsJ
To our surprise, we discovered that depending on NADPH, YtsJ switches activity from a pyruvate-producing malic enzyme to a lactate-generating malolactic enzyme. This switch in activity allows YtsJ to adaptively compensate for cellular NADPH over- and underproduction upon demand. Finally, NADPH-dependent bifunctional activity was also detected in the YtsJ homolog in
MaeB. Overall, our study extends the known redox cofactor balancing mechanisms by providing first-time evidence that the type of catalyzed reaction by an enzyme depends on metabolite abundance.
A new mechanism for NADPH balancing was discovered in
It pivots on the bifunctional enzyme YtsJ, which is known to catalyze NADP-dependent malate decarboxylation. We found that in the presence of excessive NADPH, the same enzyme switches to malolactic activity and creates a transhydrogenation cycle that ultimately converts NADPH to NADH. This provides a regulated mechanism to immediately adjust NADPH/NADP
in response to instantaneous needs.
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